We use a clean hood (like a tissue culture hood) to do ALL work with the
gels from getting them out of the glass plates to final desalted sample
loaded into the nanospray tip, except when they are concentrated. We use a
dedicated speedvac with inlet filters that is used just for in gel digests
and is cleaned every time it is used. We check the samples by maldi before
concentrating and after resuspending in 1% formic acid and desalting an
appropriate amount of the sample into 1 microliter (for low amounts, we let
this evaporate to about 0.3 microliters before adding matrix).
We desalt with nanocolumns using Poros 50. For larger proteins we desalt
in steps (5%, 10%, 20%, 30%, 40% AN). We do nanospray in Protana tips, cut
back 1 cm, and do not use the conductive paint. First we check ms at
several orifice voltages. If signal is weak, we check parent ion scans for
several immonium ions (minimally I/L) before selecting a set of peptides
for ms/ms (it helps to use the maldi spectra as guide). We run a blank for
10-15 minutes between samples (put buffer only in a needle, you can reuse
this blank all day if you load in a lot of buffer and keep it in a moist
chamber inbetween).
Katheryn Resing